The binding of the calcium channel blocker, bepridil, to calmodulin

Bepridil had the highest relative potency for inhibition of myosin light chain kinase (MLCK) activated by Ca2+-calmodulin of all the calcium channel blockers we examined. Kinetic analysis indicated that the primary effect of bepridil was mediated through a competitive inhibition of the enzyme activation by interaction with calmodulin and the apparent Ki value of this agent was 2.2 microM. We then examined the binding of bepridil to calmodulin, using the equilibrium column binding technique. [3H]bepridil bound to the calcium-calmodulin complex, but not to calmodulin in the presence of 2 mM EGTA. Scatchard analysis of the binding of bepridil to calmodulin demonstrated that the dissociation constant was 6.2 microM and the calculated number of specific binding sites was about 5 sites per molecule of calmodulin. The concentrations of unlabeled bepridil, W-7, prenylamine, verapamil and diltiazem producing 50% inhibition (IC50) of the binding of [3H]bepridil to calmodulin were 4 microM, 28 microM, 45 microM, 130 microM and 700 microM, respectively. However, nifedipine and nicardipine did not displace [3H]labeled bepridil from calmodulin. There was a good correlation between the displacement of [3H]bepridil from calmodulin and the inhibitory effect on MLCK by these calcium channel blockers and W-7. These results suggest that bepridil binds to calmodulin in the presence of calcium and potently inhibits the phosphorylation of myosin light chain.